ENGLISH ABSTRACT: Parkinson’s disease (PD) is a progressive and debilitating neurodegenerative disorder, characterized by a distinct motor phenotype and the selective loss of dopaminergic neurons in the substantia nigra. While the etiology of PD is not fully understood, it is thought to involve a combination of different genetic, cellular and environmental factors that independently or concurrently contribute to neurodegeneration. To date, several PD-causing genes have been identified, and investigations of their function have provided novel insights into the pathobiology of disease. Particularly interesting among the known PD genes is parkin, mutations in which are the most common genetic cause of early onset PD. Parkin is an E3 ligase that ubiquitinates protein substrates and targets such substrates for degradation via the ubiquitin proteasome system (UPS). Therefore, the loss of parkin may result in the deleterious accumulation or dysregulation of parkin substrates and neurotoxicity. Parkin’s enzymatic activity has also been implicated in the maintenance of mitochondrial health, and mitochondrial dysfunction is commonly reported in cellular and animal models of parkin deficiency. This study aimed to investigate parkin and its role in PD on various levels. Initially, genetic screening approaches were used to assess the contribution of parkin mutations to PD in a group of 229 South African patients. It was concluded that parkin mutations are rare in the South African PD population, being present in only seven (3.1%) patients in the study group. Interestingly, this study identified two of only three Black African PD patients with mutations in a known PD-causing gene to date. The low frequency of known PD genes raises the interesting possibility that the unique South African ethnic groups may harbor mutations in novel PD-causing genes.
Although many parkin-interacting proteins have been identified in the literature, it is anticipated that novel, pathologically-relevant parkin substrates remain to be discovered. Hence, this study used a yeast two-hybrid (Y2H) approach to identify novel parkin interactions. This yielded 29 putative parkin interactors, of which four, namely ATPAF1, SEPT9, actin and 14-3-3η, were prioritized for verification by co-localization and co-immunoprecipitation experiments. Interestingly, two of the parkin interactors (ATPAF1 and SEPT9) were found to accumulate in the absence of parkin, supporting their role as authentic parkin substrates. The identification of these two intriguing proteins implicates parkin in the regulation of mitochondrial ATP synthase assembly and septin filament dynamics, which may be of significant relevance to our understanding of processes underlying neurodegeneration. Moreover, it was aimed to assess various markers of mitochondrial function in a parkin-deficient cellular model, as previous studies had reported conflicting results regarding mitochondrial impairments in patient-derived cells with parkin mutations. Hence, dermal fibroblasts were obtained from PD patients with homozygous parkin mutations, after which cell growth and viability, mitochondrial membrane potential, respiratory rates and the integrity of the mitochondrial network were assessed. Surprisingly, it was found that cell growth was significantly higher in the parkin-mutant fibroblasts compared to wild-type controls fibroblasts under basal conditions (p=0.0001), while exhibiting a greater inhibition of cell growth in the presence of the mitochondrial toxin CCCP (p=0.0013). Furthermore, whereas the mitochondrial networks of patient-derived fibroblasts were more fragmented than controls (p=0.0306), it was found that mitochondrial respiratory rates were paradoxically higher in the patients (p=0.0355). These unanticipated findings are suggestive of a compensatory response to the absence of parkin. The parkin-deficient cellular model was also used in a pilot study of the functional effects of vitamin K2 treatment, which has recently been identified as a promising PD therapeutic modality. It was found that treatment with vitamin K2 resulted in more interconnected mitochondrial networks (p=0.0001) and enhanced respiratory rates (p=0.0459) in both parkin-mutant and wild-type control cells. While these results need to be studied further, it suggests that vitamin K2 supplementation may be of use as a general promoter of mitochondrial integrity and function.
In conclusion, this dissertation highlights some novel interactions of the parkin protein and some interesting phenotypes of parkin deficiency. It is hoped that further investigation of parkin and its role in PD will, ultimately, aid in the development of therapeutic strategies to treat this debilitating and poorly-understood disorder.